Hydrogels are a class of highly hydrated materials with 3 dimensional networks composed of hydrophilic polymer chains, which are either synthetic or natural in origin. Because they have mechanical and structural properties similar to native tissues and the extracelluar matrix, hydrogels have been widely employed as implantable medical devices, including in contact lenses and biosensors, immunoisolating capsules for tissue transplantations, scaffolds for tissue regeneration, and materials for drug delivery. Accordingly, there is a need for biocompatible hydrogels capable of deployment by minimally invasive methods and solidification under physiological conditions.
Native chemical ligation (NCL) is the reaction between a thioester moiety and a cysteine moiety with a free a-amine group to yield an S-acyl covalent intermediate that spontaneously undergoes an S- to N-acyl migration to form a new amide bond. This mild ligation method has proven useful in chemical synthesis of large peptides and proteins and dendrimers, and has been combined with peptide self-assembly to generate polypeptides with repeated sequences. NCL has been used to increase the stiffness of hydrogels pre-assembled from β-sheet forming peptide through cross-linking of terminal thioester and cysteine groups on self-assembled linear peptides.
In our previous invention (U.S. Patent Application Publication No. 2008/0274980), we reported the successful application of native chemical ligation to cross-link soluble macromonomers to form robust, functionalized hydrogels as potential 2D and 3D-cell culture devices for organ transplantation therapy and cell-based drug delivery. However, as shown in FIG. 1, our previously disclosed method of hydrogel formation by NCL, occurring between the 4-armed cysteine terminated macromonomer 1 and 4-armed thioester-terminated macromonomer 2, releases a low molecular weight, soluble by-product (ethyl 3-mercaptopropriate, structure 3) that is potentially cytotoxic to certain cell lines.
The dose-dependent cytotoxicity of this soluble by-product has been demonstrated in, e.g., the mouse-islet derived cell line MIN6, and removal of this side product during in situ hydrogel formation is essential for improving viability of cells encapsulated in such hydrogels. Removal can be accomplished using a mini-dialysis device. However, this added toxin removal step reduces the potential of using NCL-mediated hydrogel cross-linking in biomedical applications. Due to the adverse effects of free thiol by-products, the previously disclosed method may not be usable in certain applications, such as in creating injectable materials for use in surgical sutures or for local delivery of cell and drugs.
Accordingly, a need exists for a method of preparing macromonomer-based hydrogels for biomedical applications using native chemical ligation that does not produce a toxic thiol by-product.